By comparing the structures of different cyan fluorescent proteins, researchers figure out what makes some brighter than others—and how to make one even brighter.

When structural biologist David von Stetten set out to look at the 3-D
arrangement of fluorescent molecules, his goal was to answer a basic
scientific question: how do structural elements affect a molecule’s
brightness? But in the end, von Stetten discovered something else—how to
make an even brighter cyan protein.

After scientists had begun attaching green fluorescent protein (GFP) to
proteins to visualize the molecules during the 1990s, they then developed
different colored versions of the protein, like yellow (YFP) and cyan (CFP),
to label multiple targets within a single experiment.

Crystals of mTurquoise2, like the one shown above, let researchers study what makes some fluorescent molecules shine more brightly than others. Credit: von Stetten/Royant/CNRS-ESRF

“The problem with these variants was that they were all artificial, and hadn’t
gone through any rounds of natural evolution,” says von Stetten of the
European Synchrotron Radiation Facility in France. “They were altered to
change the color they emitted, but this also changed the structure of the
whole protein and made it weaker.”

So, scientists made educated guesses as to what further mutations could make
these variants brighter, leading to versions with improved quantum yields,
like enhanced cyan fluorescent protein (ECFP) and super cyan fluorescent
protein 3A (SCFP3A). If a protein’s quantum yield is 100%, it emits all the
light shone on it as fluorescence. Early versions of cyan proteins, however,
had quantum yields around 36%; the latest version—mTurquoise—has a quantum
yield of 83%. To understand how the mTurquoise’s structure improved its
quantum yield, von Stetten and colleagues crystallized different proteins in
the cyan family and looked at the arrangement of amino acids within them.

“We found some explanation why mTurquoise is brighter,” says von Stetten. “And
it mainly has to do with the overall stability of the protein’s structure.”
In a less-bright cyan fluorescent protein, floppy amino acids hit against
the chromophore—the crucial part of the protein responsible for its
color—and destroy its fluorescence. The brighter mTurquoise had mutations
that led to fewer floppy bits near the chromophore.

“What surprised us what how little changes have such big effects,” says von
Stetten. “Basically a single change in a hydrogen bond can change the
quantum yield from 36–56%.” As they analyzed mTurquoise’s structure in
further detail, they soon realized that there was still one weak spot.

So, von Stetten’s collaborators, a team of biologists at the University of
Amsterdam, replaced the weak amino acid. While 18 of the 19 possible
replacement amino acids decreased mTurquoise’s brightness, one improved it.
They dubbed the new protein mTurquoise2. Not only was it brighter than
mTurquoise, with a quantum yield of 93%, but it also performed better in
other lab testing, shining for a longer time (1).

“The science that’s done with these kinds of proteins is always at the
detection limit,” says von Stetten. “So, by increasing that detection limit
by even 10%, it opens up a whole new level of experimentation that wasn’t
possible before.”

Next, von Stetten’s team plans to tackle other fluorescent molecules, such as
the popular yellow variants, to determine if there are ways to further
stabilize their structures. “There’s a whole rainbow of colors that exists
now and so we think there’s probably room for improvement in some of the
other fluorescent proteins as well,” he says.